Study of Chronic Pelvic Pain Research Network Study HHS ...

Toll-like Receptor 4 and Comorbid Pain in Interstitial Cystitis/Bladder Pain Syndrome: A Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network Study

Andrew Schrepf1, Catherine S. Bradley2,3, Michael O'Donnell2, Yi Luo2, Steven E. Harte4, Karl Kreder2,3, Susan Lutgendorf1,2,3, and the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network1Department of Psychology, University of Iowa

2Department of Urology, University of Iowa

3Department of Obstetrics and Gynecology, University of Iowa

4 Departments of Anesthesiology and Internal Medicine-Rheumatology, University of Michigan

Abstract

Background—Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a condition characterized

by pelvic pain and urinary symptoms. Some IC/BPS patients have pain confined to the pelvic

region, while others suffer widespread pain. Inflammatory processes have previously been linked

to pelvic pain in IC/BPS, but their association with widespread pain in IC/BPS has not been

characterized.

Methods—Sixty-six women meeting criteria for IC/BPS completed self-report measures of pain

as part of the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP), collected 3

days of saliva for cortisol assays, and provided blood samples. Peripheral blood mononuclear cells

(PBMCs) were stimulated with Toll-Like Receptor (TLR) 2 and 4 agonists and cytokines were

measured in supernatant; IL-6 was also measured in plasma. Associations between inflammatory

variables and the likelihood of endorsing extra-pelvic pain, or the presence of a comorbid

syndrome, were tested by logistic regression and General Linear Models, respectively. A subset of

patients (n=32) completed Quantitative Sensory Testing.

Corresponding Author: Susan Lutgendorf, E11 Seashore Hall, Iowa City, IA 52242, 319-335-2432, [email protected].

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Financial DisclosuresDr. Harte has received consulting fees from Analgesic Solutions, DeCode genetics, and Pfizer, and has received grant support from Forest Laboratories and Cerephex, outside the submitted work; in addition, Dr. Harte has proprietary interest in the Multimodal Automated Sensory Testing system used in this research, including inventorship on United States and International patent applications, a licensing agreement, and the right to receive royalties from product commercialization. Dr. Kreder has received honorariums from Tengion and Medtronic, and served as a consultant for Tengion, Medtronic, and Symptelligence, which he has an equity interest in, outside the submitted work. Mr. Schrepf and Drs. Bradley, O'Donnell, Luo and Lutgendorf report no financial disclosures or potential conflicts of interest.

HHS Public AccessAuthor manuscriptBrain Behav Immun. Author manuscript; available in PMC 2016 October 01.

Published in final edited form as:Brain Behav Immun. 2015 October ; 49: 66–74. doi:10.1016/j.bbi.2015.03.003.

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Results—A one standard deviation increase in TLR-4 inflammatory response was associated

with a 1.59 greater likelihood of endorsing extra-pelvic pain (p = .019). Participants with

comorbid syndromes also had higher inflammatory responses to TLR-4 stimulation in PBMCs (p

= .016). Lower pressure pain thresholds were marginally associated with higher TLR-4

inflammatory responses (p = .062), and significantly associated with higher IL-6 in plasma (p = .

031).

Conclusions—TLR-4 inflammatory responses in PBMCs are a marker of widespread pain in

IC/BPS, and should be explored in other conditions characterized by medically unexplained pain.

Keywords

Inflammation; Toll-Like Receptors; Functional Somatic Syndromes; Pain; Negative Affect; Interstitial Cystitis/Bladder Pain Syndrome

1. Introduction

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a highly prevalent debilitating

chronic condition characterized by pelvic/bladder pain and urinary symptoms such as

frequency, urgency, and nocturia (Hanno et al., 2011). Additionally, IC/BPS patients have a

high prevalence of psychiatric comorbidities including depression and anxiety disorders

(Clemens et al., 2008). While some patients present with Hunner's ulcers, inflammatory

lesions found on the wall of the bladder, approximately 90% do not (Simon et al, 1997).

IC/BPS is therefore a diagnosis of exclusion, and is sometimes considered a cluster of

medically unexplained symptoms.

It has been proposed that there may be distinct subtypes of IC/BPS, as some patients appear

to experience pain and discomfort in the pelvic/bladder region only (i.e. local pain), while

others report extra-pelvic pain consistent with somatic syndromes like fibromyalgia, Irritable

Bowel Syndrome (IBS), Chronic Fatigue Syndrome (CFS), or Temporomandibular joint

disorder (TMD), suggesting a condition mediated by the central nervous system. A recent

investigation found that that comorbid IBS and CFS were present in 39% and 19% of

IC/BPS patients, respectively (Nickel et al., 2010). These findings are consistent with the

results of many studies finding high degrees of comorbidity between somatic syndromes

(Wessely et al., 1999). This suggests that there may be common physiological factors that

support global changes in central pain pathways and increased pain perception (Phillips &

Clauw, 2011). Furthermore, IC/BPS patients with comorbid somatic syndromes (e.g. CFS)

appear to be at risk of developing additional somatic syndromes in the future, suggesting a

progressive element of altered pain perception in some patients (Warren et al., 2013).

Identifying markers of pain sensitization in IC/BPS may improve early phenotyping of

vulnerable patients and lead to novel therapeutic targets with the potential to prevent disease

progression. Furthermore, identifying markers of central sensitization in chronic pain

patients may further prevent psychiatric comorbidity as chronic pain has recently been

shown to induce dysfunction in the locus coeruleus and subsequent depression and anxiety

like behaviors in an animal model (Alba-Delgado et al., 2013). Much research has been

devoted to identifying altered mechanisms of pain perception (i.e. sensitized pathways), and

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whether reliable markers of these alterations can be identified. Candidate markers include

changes in pain processing networks identified through functional magnetic resonance

imaging (fMRI), and hyperalgesia/allodyina identified by quantitative sensory testing

(QST), both of which have identified abnormal responses to stimuli in chronic pain patients,

including patients with IC/BPS (Kilpatrick et al., 2014; Ness et al., 2014). Another

promising biomarker is the inflammatory response to Toll-Like Receptor (TLR) stimulation

in peripheral immune cells, as we have recently found these responses to be associated with

heightened pelvic pain in IC/BPS (Schrepf et al., 2014).

TLRs are highly conserved receptors on sentinel immune cells that respond to both Microbe

Associated Molecular Patterns (MAMPs) and Damage Associated Molecular Patterns

(DAMPs; Hutchinson et al., 2009). We have recently reported that TLR-2 inflammatory

responses distinguish IC/BPS patients from healthy controls, and that the magnitude of

TLR-4 inflammatory responses in stimulated PBMCs are associated with the extent of

painful urinary and pelvic symptoms reported by IC/BPS patients. PBMCs have been

hypothesized to mark pain sensitization in humans since it was demonstrated that

proliferation of PBMCs incubated with morphine is strongly associated with tolerance for

noxious cold stimuli (Hutchinson et al., 2004). Additionally, we found that IC/BPS patients

had higher serum levels of Interleukin (IL)-6, a marker of systemic inflammation, and

altered diurnal cortisol patterns (Schrepf et al., 2014). These finding echo a recent

investigation that found that TLR-2 and TLR-4 inflammatory responses in PBMCs

differentiate chronic pain patients from healthy controls (Kwok et al., 2012) and other work

identifying altered TLR inflammatory responses as features of other conditions

characterized by persistent pain such as Inflammatory Bowel Disease and Rheumatoid

arthritis (Kovarik et al., 2011; Kowalski et al., 2008). However, it is unknown if

inflammatory responses in PBMCs can differentiate subtypes of painful syndromes such as

IC/BPS, particularly those characterized by pain not typically considered part of the IC/BPS

syndrome (i.e. widespread, extra-pelvic pain.).

The purpose of the current study was to determine if inflammatory processes, especially

TLR-2 and TLR-4 inflammatory responses in PBMCs, are differentially associated with

pelvic vs. extra-pelvic pain in IC/BPS. Additionally, we examined relationships between

TLR-mediated inflammation, pain intensity/interference with daily life, and pressure pain

sensitivity determined by QST. We also examined the relationship between TLR-mediated

inflammation and the presence of comorbid pain conditions in IC/BPS patients, as these

conditions are characterized by pain outside the pelvic region, and contribute substantially to

the difficulty of treating the IC/BPS syndrome (Nickel et al., 2010). In line with the results

of our earlier work and results from animal models of chronic pain, we hypothesized that

greater TLR inflammatory responses in PBMCs would be associated with pain outside the

pelvic region, increased pain sensitivity by QST, and comorbid somatic syndromes.

2. Methods

2.1 MAPP Study and Recruitment

The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) is a National

Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research

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initiative comprising several sites with the objective of characterizing the epidemiology,

symptom trajectories, phenotypes and biological correlates of chronic pelvic pain (Clemens

et al. 2014). The University of Iowa is a participating institution emphasizing biomarker

research. Participants were eligible if they were at least 18 years of age, female, not

pregnant, and reported chronic pain/pressure/discomfort associated with the bladder or

pelvic region in the preceding three months. Participants had negative urine cultures for

uropathogens. Exclusion criteria included conditions which might result in tissue damage to

areas relevant to IC/BPS symptomology (e.g. history of urethral stricture, neurological

disorder affecting the bladder or bowel). Additional information about the MAPP project,

including patient characterization, study aims, and full exclusion criteria is available

(Clemens et al., 2014; Landis et al., 2014; Schrepf et al., 2014).

2.2 Demographic and Symptom Information

The sample was composed of an expanded group of participants from a previously reported

study (Schrepf et al. 2014) who provided additional information on pain and comorbid

syndromes. In addition, a subsample completed QST. Sixty-six women provided

demographic information at the time of eligibility screening, including information about

income, education, employment, race and ethnicity. Upon study entry, participants had a

blood draw, urine collection, physical examination and completed a battery of

questionnaires relating to pain and urological symptoms. These included the Brief Pain

Inventory (BPI), a measure of pain intensity, interference with daily life, and a body map for

selection of painful areas (Cleeland & Ryan, 1994) which has previously been validated in

chronic pain populations (Tan et al., 2004). The body map was modified so that patients

could select regions where pain was experienced from a standardized form of 45 distinct

areas. Participants were also administered self-report screens to assess the presence of

comorbid somatic syndromes. These included the Rome III criteria for IBS (Drossman &

Dumitrascu, 2006), the American College of Rheumatology diagnostic criteria for

Fibromyalgia (Wolfe et al., 2010), International Chronic Fatigue Syndrome Study Group

criteria for CFS (Fukuda et al., 1994), an 8 question MAPP specific diagnostic tool for

symptoms of vulvodynia (e.g. “experience constant burning or raw feeling at the opening of

the vagina,”) and the Research Diagnostic Criteria for TMD (Dworkin et al., 2002). These

diagnostic criteria show adequate reliability and validity (Dworkin et al., 2002; Ford et al.,

2013; Komaroff et al., 1996; Wolfe et al., 2010;) excepting the criteria for vulvodynia,

which is necessarily exploratory. Additionally, participants completed the reliable and

validated Positive and Negative Affect Scale (PANAS; Watson et al. 1988). Use of non-

steroidal anti-inflammatory drugs (NSAIDs), tricyclic antidepressants, opioids, selective

serotonin/norepinephrine reuptake inhibitors (SSRI/SNRI), and pentosan polysulfate, and

duration of symptoms in years, were collected by patient self-report.

2.3 Cortisol

Salivary cortisol was collected in salivettes by participants at 3 time points (upon waking:

4-9am, afternoon: 5 pm, and bedtime: 8pm-12am) for three consecutive days prior to the

baseline visit. Samples collected outside this time frame were excluded to maintain

homogeneity. Individual waking and bed times have been demonstrated to better

approximate diurnal cortisol rhythms than scheduled collection times (Kraemer et al., 2006).

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Participants were instructed not to eat, exercise or consume caffeine for thirty minutes prior

to collecting a sample. Participants wrote the time of each collection on the salivette tubes.

Self-report of collection time has been demonstrated to be reliable and salivary cortisol is

stable at room temperature (Kraemer et al., 2006). Salivettes were analyzed by

chemiluminescence immunoassay (IBL, Hamburg, Germany) at the Technical University of

Dresden. The lower detection limit is 0.41 nmol/L and inter-assay and intra-assay

coefficients of variance are less than 10%.

2.4 Inflammatory Measures

Blood samples were collected between approximately 11:30am and 12:30pm. PBMCs were

separated by Ficoll-paque gradient centrifugation within 30 minutes of blood collection and

cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin and

100 ug/ml streptomycin for 3 days with TLR agonists at 37°C in a humidified incubator

with 5% CO2. TLR-2 and TLR-4 agonists were selected on the basis of the role of these

receptors in chronic pain in animal models and our previous findings linking them to

symptoms in IC/BPS (Bastos et al., 2013; Hutchinson et al., 2008; Schrepf et al., 2014;). For

stimulation of TLR-4, 50 ng/ml of Lipopolysaccharide (LPS) was used; for TLR-2

stimulation, 0.04 ng/ml of Staphylococcus aureus Cowan I (SAC) was used. Conditioned

media was then harvested and frozen at −80°C prior to batch ELISA analysis. Each well

contained 1×106 cells in 24 well plates, with one well per subject for TLR-4 and TLR-2

stimulation. Cytokines IL-6 and IL-1β were assayed in duplicate by DuoSet ELISAs (R&D

Systems) according to instructions included with the kit. Plasma IL-6 was assayed with a

high sensitivity Quantikine ELISA (R & D Systems).

2.5 Quantitative Sensory Testing

A subsample of participants completed the MAPP network QST protocol. Pressure pain

sensitivity was evaluated at the thumbnail (As-Sanie et al., 2013; Giesecke et al., 2004;

Petzke et al., 2001) using the Multimodal Automated Sensory Testing (MAST) system

(Harte et al., 2013). The MAST system consists of a control computer that executes testing

algorithms and stores testing data, and a touch-screen interface for participant feedback.

Computer-controlled pressure stimuli are applied to the thumbnail bed via a 1 cm2 rubber

probe housed within a wireless, pistol-grip style handset. Probe movement is driven by a

miniature servo-motor. A closed-looped control system measures applied pressures and

dynamically self-adjusts motor output to the resistance of the thumb and any movement to

ensure accurate and repeatable force delivery.

Participants received scripted instructions, and MAST system familiarization and practice

testing prior to data collection. During testing, an ascending series of 5-s duration pressures

were delivered at a rate of 4 kg/cm2/s to the dominant thumbnail beginning at 0.50 kg/cm2

and increasing in 0.50 kg/cm2 steps, with a minimum inter-stimulus interval of 20 s. Pain

intensity was rated after each stimulus on a 0-100 numerical rating scale (NRS) displayed on

the interface screen (0 = no pain; 100 = worst pain imaginable). Testing terminated when the

first of three possible stop conditions were met: 1) participant reached her personal pain

tolerance (i.e., requested to stop the test), 2) patient reported a pain intensity rating of ≥

80/100, or 3) the maximum pressure of 10 kg/cm2 was delivered. A modified three-

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parameter logistic model was used to fit the stimulus-response data obtained from this

procedure. The midpoint between the minimum and maximum stimulus intensity was

estimated within-person using the SAS NLIN procedure to derive an overall measure of

supra-threshold pressure pain sensitivity, referred to as Pain50. Additional outcome

variables included pressure pain threshold, defined as the first pressure in a string of at least

two consecutive pressures that elicited a NRS pain rating > 0, and pressure pain tolerance,

defined as the last pressure recorded in the stimulus response profile.

2.6 Data Analysis

Statistical analyses were performed using SPSS v. 21 and R v. 3.1.1. Cytokine values were

log-10 transformed, and cortisol values natural log transformed, to normalize their

distribution. The Inflammatory response scores for stimulated cytokines were calculated by

summing the z-scores ([individual score- group mean]/ group standard deviation) for the

IL-6 and interleukin-1 beta (IL-1β) response in PBMCs following stimulation with LPS

(TLR-4) or SAC (TLR-2. This inflammatory response score was then standardized for ease

of interpretation. Both IL-6 and IL-1β have been implicated in enhanced pain processing

when released by spinal glia, and both are released following TLR-2 and 4 stimulation, in

part, by transcription of nuclear factor-kappaB (NFκB; Milligan & Watkins, 2009). The

composite score, therefore, is likely more reflective of the inflammatory response to TLR

stimulation than either cytokine alone. Distributions of transformed variables were examined

for confirmation of normality. Salivary cortisol values at each of the collection points were

regressed on the time of collection over the three-day period to calculate cortisol slope, a

measure of the average hourly decrease in cortisol over the course of the day as described

previously (Kraemer et al., 2006).

To determine if inflammatory variables were associated with a greater likelihood of

endorsing extra-pelvic sites as painful, mixed-effects logistic regression models were used.

Higher probabilities of endorsing pain outside the pelvic region reflect pain not typically

considered part of the IC/BPS syndrome. Thus, endorsement of pain at any of the 44 sites

outside the pelvic region was considered indicative of extra-pelvic pain. In these models the

dependent variable of interest was the probability of a patient selecting any extra-pelvic site

(44 sites) as painful, with inflammatory variables as predictors. Subject and site specific

intercepts were tested as random effects, with the maximum random effects structure

retained by likelihood testing. Modeling subject specific variance (e.g. if some patients are

more likely to endorse any site as painful) and site-specific variance (e.g. if lower back pain

is more likely to be endorsed than pain in the hands across subjects) can allow more accurate

estimation of fixed effects (Baayen et al., 2008). Random intercept terms were retained for

subject and pain site. BMI, age, use of medications, presence of a comorbid condition, and

inflammatory variables were used in univariate analyses to determine which, if any

variables, were associated with a greater likelihood of endorsing extra-pelvic pain.

Significant variables (p < .05) were retained in multivariate analyses. Relationships between

pain severity/interference (from the BPI) and inflammation were tested in multivariate

General Linear Models with the same set of covariates. To explore the relationships between

inflammatory variables (e.g. TLR-4 inflammatory responses and cortisol slope) and duration

of symptoms in years Pearson correlations were used.

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Group differences between IC/BPS only and IC/BPS comorbid patients (IBS, CFS,

fibromyalgia, TMD, vulvodynia) with respect to inflammatory variables were tested with

one-way ANOVAs, and between IC/BPS only and IC/BPS comorbid patients with

individual conditions (IBS, fibromyalgia, CFS, TMD and vulvodynia). The association

between number of comorbid conditions and inflammatory variables was tested by

Spearman's Rank correlations. Relationships between inflammatory variables and pain

intensity and interference were assessed using General Linear Models controlling for

negative affect, comorbid condition status, and use of SSRI/SSNIs, following the results of

the univariate analyses. Due to the non-normal distribution of QST Spearman's rank

correlation tests were used to test the association with inflammatory variables.

3. Results

3.1 Demographic Characteristics and Covariates

Participants were on average approximately 42 years old (range 20-74), and the vast

majority were non-Hispanic and white. See Table 1. Higher TLR-4 inflammatory responses

were associated with a greater likelihood of endorsing extra-pelvic pain (p= .006). SSRI/

SNRI use was associated with a greater likelihood of endorsing extra-pelvic pain (p < .001).

Tricyclic antidepressant use was marginally associated with a lower likelihood of endorsing

extra-pelvic pain (p = .081) whereas older age was marginally associated with a greater

likelihood of endorsing extra-pelvic pain (p =.071). Greater negative affect and presence of a

comorbid condition were both associated with a greater likelihood of endorsing extra-pelvic

pain (both p <.001). BMI, opioid use, NSAID use, Pentosan Polysulfate use, cortisol slope,

and duration of symptoms in years were not associated with the likelihood of endorsing

extra-pelvic pain (all p > .15; univariate analyses for covariate selection not shown).

Additionally, the inflammatory response to TLR-2 stimulation was not associated with a

greater likelihood of endorsing pain outside the pelvic region (Odds Ratio = 1.03, 95% CI

= .72, 1.49, p = .86). Therefore, multivariate analyses included comorbid status, SSRI/SNRI

use, and negative affect in addition to the TLR-4 inflammatory response.

3.2 TLR-4 Inflammatory Response and Pain

In multivariate analyses, a one standard deviation increase in the TLR-4 inflammatory

response was associated with 1.59 greater odds (95% CI = 1.08, 2.33) of endorsing pain

outside the pelvic area on the body map (p = .019), controlling for negative affect, comorbid

status, and SSRI/SNRI use. This one standard deviation increase in TLR-4 inflammatory

response corresponds to a 63% increase in the likelihood of a participant endorsing pain

outside the pelvic region at any site on the body map.1 See Table 2. Figure 1 illustrates the

higher likelihood of endorsing extra-pelvic pain among those with higher TLR-4

inflammatory response. The TLR-4 inflammatory score was associated with greater self-

report of pain severity (p = .013) and pain interference (p = .046) on the BPI, controlling for

negative affect, comorbid status, and SSRI/SNRI use. See Table 3. Additionally, the TLR-4

1As a conservative measure, an additional analysis was conducted including tricyclic antidepressant use and patient age in addition to the above mentioned covariates. Neither tricyclic antidepressant use nor patient age were significantly associated with extra-pelvic pain (both p > .08) and their inclusion did not attenuate the association between TLR-4 mediated inflammation and extra-pelvic pain (odds ratio: 1.63, p = .013).

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inflammatory response was associated with higher TLR-2 inflammatory responses (r = .304,

p = .013) and longer duration of symptoms in years (r = .295, p=.017) but not with cortisol

slope (r = .165, p = .23 (n=56) or plasma IL-6 (r = .064, p = .61).

3.3 QST

In the subsample of 32 patients (13 IC/BPS only, 19 IC/BPS comorbid) who underwent

QST, higherTLR-4 inflammation scores were marginally associated with lower pressure

pain thresholds (Spearman's Rho= −.334, p = .062) and increased levels of IL-6 in plasma

were significantly associated with lower pressure pain thresholds (Spearman's Rho = −.381,

p = .031). In contrast, the TLR-2 inflammation score was not associated with pressure pain

thresholds (p =.30), nor was cortisol slope (p = .84). Pain50 and pressure pain tolerance

were not associated with inflammatory variables (all p > .19).

3.4 Inflammatory Measures and Comorbid Conditions

The TLR-4 inflammatory response was significantly greater in IC/BPS comorbid patients (p

= .016) compared to the IC/BPS patients without comorbid conditions. Additionally, a

higher TLR-4 inflammatory response distinguished IC/BPS + IBS patients (n=28, p = .018),

IC/BPS + TMD (n=23, p = .007), and IC + vulvodynia (n=14, p = .049), from IC/BPS only

participants (n=26). The TLR-4 inflammatory response was higher in IC/BPS + CFS

participants (n=8) and IC/BPS + fibromyalgia participants (n=2) but not significantly so

(both p > .18), possibly due to small sample sizes. IL-1β measured in supernatant of LPS

(TLR-4) stimulated PBMCs was significantly higher in IC/BPS patients with comorbid

conditions (p = .008) whereas IL-6 was not significantly elevated (p = .21). IL-1β and IL-6

measured in the supernatant of SAC (TLR-2)-stimulated PBMCs did not differ significantly

between groups (both p > .20) nor did the TLR-2 composite inflammatory score (p = .50).

IL-6 measured in plasma was marginally higher in IC/BPS comorbid participants (p = .097,

while cortisol slopes did not differ between groups (p = .96). See Table 4. Greater numbers

of comorbid syndromes were associated with a greater TLR-4 inflammatory response

(Spearman's Rho = .311, p = .011). No other inflammatory variable was associated with

number of comorbid conditions or presence of individual comorbid conditions (all p >.10).

4.1 Discussion

The key finding of this study is that TLR-4-mediated inflammatory responses in PBMCs are

associated with extra-pelvic pain in a chronic pelvic pain population. This is demonstrated in

the association between LPS evoked inflammation and an increasing likelihood of endorsing

pain outside the pelvic region, and by the ability of this inflammatory response to distinguish

between patients meeting criteria for IC/BPS only and patients meeting criteria for IC/BPS

who also had comorbid syndromes. Use of medications was not associated with measures of

pain, while greater negative affect was strongly associated with pain measures independent

of TLR-4 inflammation. While exploratory, QST data suggest that TLR-4 inflammation may

also be associated with lower pressure pain thresholds measured at a non-symptomatic site

remote from the pelvic region (i.e., the thumb), further suggesting a central mechanism of

pain hypersensitivity in this population. These findings build on our previous finding that

TLR-4 inflammation is associated with pelvic pain in IC/BPS (Schrepf et al., 2014) by

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demonstrating that TLR-4 inflammation is associated with comorbid pain not typically

considered part of the IC/BPS syndrome.

This is the first study to our knowledge which has shown TLR-mediated inflammation to be

associated with comorbid pain in a chronic pain population. Further, these differences in

TLR-4 mediated inflammation were not associated with a particular comorbid condition, as

each condition that was well-represented in our sample (IBS, TMD, and vulvodynia) was

characterized by higher TLR-4 mediated inflammation compared to patients with IC/BPS

only. This suggests that TLR-4 inflammation may reflect a broad mechanism by which pain

signaling is enhanced in IC/BPS. This extends our earlier finding that TLR-4 inflammation

predicts non-specific pain severity and frequency of pelvic pain symptoms in IC/BPS but

was not associated with pain in particular anatomical regions or during particular activities.

A recent investigation in an animal model of visceral pain found that TLR-4 regulates stress-

induced pain (Tramullas et al., 2014). This is notable given the high prevalence of IBS in

IC/BPS patients, and because stressful events frequently precipitate symptom flares

(Rothrock et al., 2001). QST data indicating lower pressure pain thresholds (increased pain

sensitivity) in patients with higher TLR-4 inflammatory responses and higher IL-6 in blood

suggest that these inflammatory processes are related to global pain sensitivity, not pain

associated with IC/BPS only, and are consistent with other findings that higher pro-

inflammatory cytokines in blood are associated with altered pain sensitivity on QST in

osteoarthritis patients (Lee et al., 2011). While the presence of a comorbid syndrome was

associated with an increased likelihood of endorsing extra-pelvic pain in a univariate model,

this was no longer true in the multivariate model including TLR-4 inflammation and

negative affect. This provides further evidence that TLR-4 inflammation may play a critical

role in the painful symptoms associated with comorbid conditions in IC/BPS, though it is

not possible to determine from these results what role, if any, TLR-4 inflammation may play

in centrally mediated pain sensitization.

The fact that TLR-2 stimulation distinguished patients from comparison participants in our

previous study, but did not distinguish between patients with and without comorbid

syndromes, is of interest. One possibility is that hypersensitivity to TLR-2 is an early or

universal feature of IC/BPS while TLR-4 sensitivity is not. Previous research has

demonstrated that higher TLR-2 density on PBMCs, but not TLR-4 density, distinguishes

IBS patients from controls (Ohman et al., 2012). Another recent study found that TLR-2 and

TLR-4 mRNA were up-regulated in the colonic mucosal tissue of IBS patients with

heterogeneous symptom presentation compared to those with symptoms of diarrhea or

constipation only (Belmonte et al. 2012). It is unknown if TLR-4 density on PBMCs or other

tissue may differ between IC/BPS patients with and without comorbid conditions, or if

TLR-4 responses are heightened in patients with comorbid syndromes due to a “priming”

effect of prior MAMP or DAMP exposure. Another possibility is that intracellular signaling

and subsequent cytokine production may differ significantly following TLR-4 vs. TLR-2

stimulation; a recent investigation of stimulated human whole blood found that both TLR-2

and TLR-4 stimulation resulted in NF-κB family activity, but that a distinct IFN

upregulation occurred following TLR-4 stimulation only (Blankley et al., 2014). Another

recent investigation used principal component analysis to analyze various cytokine and

chemokine responses to both TLR-2 and TLR-4 receptor stimulation in human whole blood;

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the results indicated that different TLR agonists (including TLR-2 and TLR-4), evoked

distinct protein signatures, suggesting divergent intracellular signaling pathways (Duffy et

al., 2014). Thus it appears that though TLR2 and 4 ligands may signal through the same

receptor, each cytokine is capable of eliciting unique signaling patterns. Another possibility

is that unexplored vulnerabilities (e.g. genetic factors) could mask the relationship between

TLR-2 mediated inflammation and pain, if such a relationship exists. Clearly, more research

is needed to identify relevant cellular and intracellular differences in TLR-4 vs. 2 responses

in the context of IC/BPS.

Microglia and astrocytes express TLRs including TLR-2 and TLR-4, and stimulation of

TLR-4 on microglia induces release of pro-inflammatory cytokines IL-6, IL-1β- and TNF-

alpha in the spinal cord (Milligan & Watkins, 2009). TLR-4-mediated inflammation released

by glia cells in the dorsal horn of the spinal cord is thought to be one contributing

mechanism for central pain amplification in rodent models of chronic pain (Ellis et al., 2014;

Grace et al., 2014; Hutchinson et al., 2008), though this effect may be sex specific, as recent

work suggests that LPS promotes hyperalgesia when delivered to the brain or periphery, but

not the spinal cord, in female mice (Sorge et al. 2011). Regardless, TLR-4 is essential in

LPS induced hyperalgesia as LPS injection fails to promote hyperalgesia in TLR-4 deficient

mice (Mattioli et al., 2014). Importantly for this study, the amplification of pain signaling

sometimes involves extension of pain from the original site of injury, termed “extra-

territorial” pain (Wieseler-Frank et al., 2005). Whether circulating PBMCs reflect neuro-

inflammatory processes remains an open question; recent work suggests that TLR-mediated

inflammation in PBMCs corresponds to the same TLR-mediated inflammation in the spinal

cord, in a rodent model of chronic pain (Kwok et al., 2013). However, relevant animal

models of IC/BPS will need to be developed before concordance between inflammatory

responses in PBMCs and spinal microglia can be formally tested.

While rodent models that investigate the role of TLRs in pain have typically used

neuropathic injury models (e.g. sciatic constriction), studies of human pain populations with

little evidence of peripheral tissue damage have also identified differential responses to TLR

stimulation in PBMCs (Kowalski et al., 2008; Schrepf et al., 2014). If sensitization of TLR-

induced inflammation is a mechanism for pain amplification in IC/BPS, this raises the

question of what the initiating events may be in this population, given the generally low

proportion of patients with evidence of peripheral tissue damage. One large twin study

implicated both genetic factors (approximately one third) and non-shared environmental

factors (approximately two thirds) in the risk of IC/BPS (Altman et al., 2011). At least two

large studies have identified an increased number of antecedent urogenital infections as a

risk factor for IC/BPS in women raising the possibility that recurrent or severe infections

might serve as initiating events in IC/BPS (Díaz Mohedo et al., 2014; Li et al. 2010). TLRs

including TLR-4 are pattern recognition receptors that respond to PAMPs and DAMPs; they

play a critical role in expelling bacteria from the urinary tract and are expressed on both

bladder epithelial cells and phagocytic cells that migrate into the bladder during infections

(Song & Abraham, 2008). Purified LPS infused directly into the bladder induces pain in the

pelvic region in a rodent model of urinary tract infection (Rudick et al., 2010). One

possibility, therefore, is that sustained local inflammatory events precipitate TLR

sensitization in migrating immune cells that then begin to modulate central pain processing

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after evidence of local infection is gone. This concept is supported by experiments

demonstrating that a single peripheral inflammatory challenge (e.g. formalin) or peripheral

trauma (e.g. laparotomy) can prime spinal microglia activation and subsequent LPS induced

allodynia for as long as two weeks (Hains et al., 2010). More direct implications for IC/BPS

symptoms have been demonstrated in an animal study that found a demyelination injury to

the sciatic nerve increased the sensitivity of bladder-associated sensory neurons to

chemokines and increased the frequency of micturition (Foster et al., 2011).

4.2 Limitations

This sample of IC/BPS patients was disproportionately non-Hispanic and white compared to

the general population. These findings require replication in a more diverse sample. Isolated

PBMCs were stimulated at a single dose-level of LPS and SAC. It is possible that

characterizing TLR-2 inflammatory responses at a wide range of doses might reveal

differences between IC/BPS only patients and those suffering comorbid conditions (Kwok et

al., 2013). These analyses are cross-sectional and cannot determine causal directions

between pain measures and TLR-mediated inflammation. A large number of statistical tests

were performed in exploring these novel hypotheses; these associations require replication

in other samples of IC/BPS patients.

4.1 Conclusions and Future Directions

TLR-4 mediated inflammation is a promising biomarker of comorbid pain in IC/BPS

patients and may be associated with pain in other somatic syndromes. Putative TLR-4

antagonists that have been shown effective at suppressing pain in animal models may have

application in IC/BPS populations. In addition to characterizing TLR-mediated

inflammatory responses at a wider range of concentrations, more research is required to

delineate the relationship between local inflammatory events and central pain sensitivity. As

IC/BPS is often characterized by symptom fluctuation, termed “flares,” future studies should

consider TLR-mediated inflammation in relation to these events.

Acknowledgments

The authors gratefully acknowledge the assistance of Mary Eno in carrying out this project. This research was funded by grant UO1DK082344 to K.K. from the National Institute of Diabetes Digestive and Kidney Diseases and by the Institute for Clinical and Translational Science at the University of Iowa, grant 2 UL1 TR000442-06 . The authors gratefully acknowledge the MAPP research network:

MAPP Network Executive Committee: J. Quentin Clemens, MD, FACS, MSci, Network Chair, 2013-, Philip Hanno, MD, Ziya Kirkali, MD, John W. Kusek, PhD, J. Richard Landis, PhD, M. Scott Lucia, MD, Chris Mullins, PhD, Michel A. Pontari, MD. Northwestern University Discovery Site : David J. Klumpp, PhD, Co-Director, Anthony J. Schaeffer, MD, Co-Director, Apkar (Vania) Apkarian, PhD, David Cella, PhD, Melissa A. Farmer, PhD, Colleen Fitzgerald, MD, Richard Gershon, PhD, James W. Griffith, PhD, Charles J. Heckman II, PhD, Mingchen Jiang, PhD, Laurie Keefer, PhD, Darlene S. Marko, RN, BSN, CCRC, Jean Michniewicz, Todd Parrish, PhD, Frank Tu, MD, MPH. University of California, Los Angeles Discovery Site and PAIN Neuroimaging Core: Emeran A. Mayer, MD, Co-Director, Larissa V. Rodríguez, MD, Co-Director, Jeffry Alger, PhD, Cody P. Ashe-McNalley, Ben Ellingson, PhD, Nuwanthi Heendeniya, Lisa Kilpatrick, PhD, Jason Kutch, PhD, Jennifer S. Labus, PhD, Bruce D. Naliboff, PhD, Fornessa Randal, Suzanne R. Smith, RN, NP. University of Iowa Discovery Site: Karl J. Kreder, MD, MBA, Director, Catherine S. Bradley, MD, MSCE, Mary Eno, RN, RA II, Kris Greiner, BA, Yi Luo, PhD, MD, Susan K. Lutgendorf, PhD, Michael A. O’Donnell, MD, Barbara Ziegler, BA. University of Michigan Discovery Site: Daniel J. Clauw, MD, Co-Director; Network Chair, 2008-2013, J. Quentin Clemens, MD, FACS, MSci, Co-Director; Network Chair, 2013-, Suzie As-Sanie, MD, Sandra Berry, MA, Megan E. Halvorson, BS, CCRP, Richard Harris, PhD, Steve Harte, PhD, Eric Ichesco, BS, Ann Oldendorf, MD, Katherine A. Scott, RN, BSN, David A. Williams, PhD. University of Washington, Seattle Discovery Site: Dedra Buchwald, MD, Director,

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Niloofar Afari, PhD, Univ. Of California, San Diego, John Krieger, MD, Jane Miller, MD, Stephanie Richey, BS, Susan O. Ross, RN, MN, Roberta Spiro, MS, TJ Sundsvold, MPH, Eric Strachan, PhD, Claire C. Yang, MD. Washington University, St. Louis Discovery Site: Gerald L. Andriole, MD, Co-Director, H. Henry Lai, MD, Co-Director, Rebecca L. Bristol, BA, BS, Coordinator, Graham Colditz, MD, DrPH, Georg Deutsch, PhD, Univ. of Alabama at Birmingham, Vivien C. Gardner, RN, BSN, Coordinator, Robert W. Gereau IV, PhD, Jeffrey P Henderson, MD, PhD, Barry A. Hong, PhD, FAACP, Thomas M. Hooton, MD, Univ of Miami, Timothy J. Ness, MD, PhD, Univ. of Alabama at Birmingham, Carol S. North, MD, MPE, Univ. Texas Southwestern, Theresa M. Spitznagle, PT, DPT, WCS, Siobhan Sutcliffe, PhD, ScM, MHS. University of Pennsylvania Data Coordinating Core (DCC): J. Richard Landis, PhD, Core Director, Ted Barrell, BA, Philip Hanno, MD, Xiaoling Hou, MS, Tamara Howard, MPH, Michel A. Pontari, MD, Nancy Robinson, PhD, Alisa Stephens, PhD, Yanli Wang, MS, University of Colorado Denver Tissue Analysis & Technology Core (TATC): M. Scott Lucia, MD, Core Director, Adrie van Bokhoven, PhD, Andrea A. Osypuk, BS, Robert Dayton, Jr, Karen R. Jonscher, PhD, Holly T. Sullivan, BS, R. Storey Wilson, MS. Drexel University College of Medicine: Garth D.Ehrlich, PhD. Harvard Medical School/Boston Children's Hospital: Marsha A. Moses, PhD, Director, Andrew C. Briscoe, David Briscoe, MD, Adam Curatolo, BA, John Froehlich, PhD, Richard S. Lee, MD, Monisha Sachdev, BS, Keith R. Solomon, PhD, Hanno Steen, PhD. Stanford University : ,Sean Mackey, MD, PhD, Director, Epifanio Bagarinao, PhD, Lauren C. Foster, BA, Emily Hubbard, BA, Kevin A. Johnson, PhD, RN, Katherine T. Martucci, PhD, Rebecca L. McCue, BA, Rachel R. Moericke, MA, Aneesha Nilakantan, BA, Noorulain Noor, BS. Queens University: J. Curtis Nickel, MD, FRCSC, Director. National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH): Chris Mullins, PhD, John W. Kusek, PhD, Ziya Kirkali, MD, Tamara G. Bavendam, MD

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Highlights

• We measured inflammatory responses in PBMCs to TLR stimulation in IC/BPS

patients

• Greater responses to TLR-4 stimulation were associated with widespread pain

• Greater responses to TLR-4 stimulation were associated with comorbid

conditions

• TLR-4 mediated inflammation may be a therapeutic target in unexplained

chronic pain

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Figure 1. Prevalence of pain reported in the first tertile (low) of the TLR-4 composite inflammatory

score versus the third tertile (high) for each site on the body map.

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Table 1

Participant Characteristics.

Participant Characteristics N=66

Age Mean(SD) 42.03 (15.12)

BMI Mean(SD) 27.18 (5.65)

PANAS negative affect 22.27 (8.56)

Race % (n)

White 97 (64)

Asian 1 (1)

Multi Race 1 (1)

Ethnicity % (n)

Non-Hispanic 98 (65)

Hispanic 1 (1)

Education % (n)

High School or GED 13 (9)

Some College 27 (18)

Graduated College 32 (21)

Graduate Degree 27 (18)

Employment % (n)

Employed 62 (41)

Unemployed 11 (7)

Disabled 8 (5)

Retired 9 (6)

Full Time Homemaker 9 (6)

Not Answered 1 (1)

Annual Income/$ % (n)

<10,000 14 (9)

<25,000 6 (4)

<50,000 23 (15)

<100,000 33 (22)

>100,000 20 (13)

Prefer not to answer 5 (3)

Comorbid Conditions % (n)

None 39 (26)

Irritable Bowel Syndrome 42 (28)

Fibromyalgia 3 (2)

Chronic Fatigue Syndrome 12 (8)

TMD 35 (23)


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Participant Characteristics N=66

Vulvodynia 21 (14)

Number of Comorbid Conditions % (n)

0 39 (26)

1 26 (17)

2 23 (15)

3 6 (4)

4 6 (4)

Tricyclic anti-depressants

No 56 (37)

Yes 44 (29)

Opioids

No 83 (55)

Yes 17 (11)

Pentosan Polysulfate

No 56 (37)

Yes 44 (29)

NSAIDs

No 89 (59)

Yes 11 (7)

SSRI/SNRIs

No 86 (57)

Yes 14 (9)

IC/BPS=Interstitial Cystitis/Bladder Pain Syndrome. BMI=Body Mass Index. NSAID= non-steroidal anti-inflammatory drug. SSRI/SNRI = selective serotonin reuptake inhibitor/serotonin-norepinephrine reuptake inhibitor. TMD = temporomandibular disorders.


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Table 2

Relationship of TLR-4 inflammatory response and covariates (logistic regression) with extra-pelvic pain.

Outcome Measure Predictor Est. S.E. Z value Odds Ratio 95% CI p

Extra-Pelvic Pain

TLR-4 inflammation response (one SD above mean) .46 .20 2.34 1.591.08, 2.33

.019

Use of SSRI/SNRI .41 .57 .73 1.51.50, 4.59

.47

Comorbid Condition .39 .43 .92 1.48.64, 4.59

.36

PANAS negative affect (one SD above mean) .66 .21 3.12 1.94, 1.28, 2.95 .002

BPI= Brief Pain Inventory. IL-6=Interleukin-6. PANAS=Positive and Negative Affect Scale. TLR=Toll-Like Receptor.


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Table 3

General Linear Models testing relationship of TLR-4 inflammatory response and covariates with BPI

symptom scores.

Outcome Measure Predictor B. S.E. F1,65 95% CI p

Pain Severity (BPI)

TLR-4 inflammatory response .503 .197 6.53 .109,.896 .013

Comorbid condition .204 .429 .23 −1.063, .655 .64

Use of SSRI/SNRI −.156 .615 .06 −1.075, 1.386 .97

PANAS negative affect .096 .025 14.83 .046,.145 <.01

Pain Interference (BPI)

TLR-4 inflammatory response .567 .278 4.17 .012, 1.123 .046

comorbid condition .200 .607 .50 −1.414, 1.014 .74

Use of SSRI/SNRI −.028 .832 .01 −1.635, 1.691 .97

PANAS negative affect .171 .035 23.67 .101,.241 <.01

BPI= Brief Pain Inventory. IL-6=Interleukin-6. PANAS=Positive and Negative Affect Scale. TLR=Toll-Like Receptor.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Schrepf et al.

Table 4

Means, standard deviations and 95% confidence intervals of biomarkers in participants with IC/BPS only and

those with additional comorbid conditions (Fibromyalgia, Irritable Bowel Syndrome, vulvodynia, Chronic

Fatigue Syndrome, Temperomandibular Disorder)

Variable Mean (S.D.) 95% CI Inflammatory Variables IC Only n=26 IC Comorbid n=40 p

TLR-4 inflammatory response −.36 (1.13)−.82, .09

.24 (.85)−.03, .51

.016

IL-1β + LPS 3.11 (.93)2.73,3.48

3.58 (.44)3.44, 3.72

.008

IL-6 + LPS 4.15 (.40)3.99, 4.31

4.28 (.42)4.14, 4.41

.205

TLR-2 inflammatory response −.10 (1.06)−.53, .32

.07 (.97)−.24, .38

.501

IL-1β + SAC 1.17 (1.19).69, 1.65

1.44 (1.14)1.08, 1.81

.365

IL-6 + SAC 1.68 (1.37)1.13, 2.24

1.79 (1.22)1.40, 2.19

.719

Cortisol Slope (ln transformed) n=22−.11 (.06)−.14, −.08

n=34−.11 (.07)−.13, −.08

.955

IL-6 plasma (log10 transformed) .33 (.35)−.75, 0.84

.46 (.26).00, .97

.097

Duration of Symptoms (years) 6.91 (7.48)3.89, 9.93

8.3 (8.00)5.71, 10.90

.48

IL-6=Interleukin-6. IL-1β=Interleukin-1 beta. LPS= lipopolysaccharide. SAC= Staphylococcus aureus Cowan. TLR = Toll-Like Receptor.


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